Báo cáo y học: " Regulation of HIV-1 transcription in cells of the monocyte-macrophage lineage" docx

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Retrovirology
Open Access
Review
Regulation of HIV-1 transcription in cells of the
monocyte-macrophage lineage
Evelyn M Kilareski
1,2,3
, Sonia Shah
1,2,3
, Michael R Nonnemacher
1,2,3
and
Brian Wigdahl*
1,2,3
Address:
1
Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel
University College of Medicine, 245 N 15th St, Philadelphia, Pennsylvania 19102, USA,
2
Center for Molecular Therapeutics and Resistance,
Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, 245 N 15th St, Philadelphia, Pennsylvania 19102,
USA and
3
Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, Pennsylvania
19129, USA
Email: Evelyn M Kilareski - ; Sonia Shah - ;
Michael R Nonnemacher - ; Brian Wigdahl* -
* Corresponding author

Abstract
Human immunodeficiency virus type 1 (HIV-1) has been shown to replicate productively in cells of
the monocyte-macrophage lineage, although replication occurs to a lesser extent than in infected
T cells. As cells of the monocyte-macrophage lineage become differentiated and activated and
subsequently travel to a variety of end organs, they become a source of infectious virus and
secreted viral proteins and cellular products that likely initiate pathological consequences in a
number of organ systems. During this process, alterations in a number of signaling pathways,
including the level and functional properties of many cellular transcription factors, alter the course
of HIV-1 long terminal repeat (LTR)-directed gene expression. This process ultimately results in
events that contribute to the pathogenesis of HIV-1 infection. First, increased transcription leads
to the upregulation of infectious virus production, and the increased production of viral proteins
(gp120, Tat, Nef, and Vpr), which have additional activities as extracellular proteins. Increased viral
production and the presence of toxic proteins lead to enhanced deregulation of cellular functions
increasing the production of toxic cellular proteins and metabolites and the resulting organ-specific
pathologic consequences such as neuroAIDS. This article reviews the structural and functional
features of the cis-acting elements upstream and downstream of the transcriptional start site in the
retroviral LTR. It also includes a discussion of the regulation of the retroviral LTR in the monocyte-
macrophage lineage during virus infection of the bone marrow, the peripheral blood, the lymphoid
tissues, and end organs such as the brain. The impact of genetic variation on LTR-directed
transcription during the course of retrovirus disease is also reviewed.
Introduction
Approximately 33.2 million people are infected with the
human immunodeficiency virus type 1 (HIV-1) world-
wide, including 2.5 million people who were newly
infected in 2007 [1]. Although fewer people are currently
infected with HIV type 2 (HIV-2), this virus is spreading
from its origin in West Africa to the Americas, Asia, and
Europe [2] and reviewed in [3-5]). In addition to being
Published: 23 December 2009
Retrovirology 2009, 6:118 doi:10.1186/1742-4690-6-118

Received: 9 July 2009
Accepted: 23 December 2009
This article is available from: />© 2009 Kilareski et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( />),
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Retrovirology 2009, 6:118 />Page 2 of 24
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the causative agent of the acquired immunodeficiency
syndrome (AIDS), HIV-1 can cause neurological prob-
lems, ranging in severity from minor cognitive/motor dys-
function (MCMD) to HIV-1-associated dementia (HAD)
(reviewed in [6-9]).
Cells of the monocyte-macrophage lineage play an impor-
tant role in the transmission and pathogenesis of HIV [10-
12]. When transmission occurs vaginally, rectally, or
orally, the primary cells involved in the transmission
event are dendritic cells [13]. However, during mucosal
trauma, inflammation, and ulceration, the epithelial bar-
rier may be disrupted and provide HIV with direct access
to the mucosal microcirculation and/or provide direc-
tional signals to recruit highly susceptible, activated,
inflammatory monocytes and T cells [14]. Circulating
monocytes can also be infected and then migrate to
peripheral tissues, including the brain [15,16], lung [17],
lymphatic system [18], bone marrow [19,20], and kidney
(reviewed in [21]). Infected monocytes can differentiate
into monocyte-derived macrophages (MDMs) and may
form a long-lived reservoir for the virus [22-25]. Addition-
ally, MDMs can be infected after differentiation and are
more susceptible to new infection in comparison to

freshly isolated monocytes due to increased expression of
the HIV co-receptor CCR5 [26]; however, this infection is
limited, and the production of virus is hindered at many
steps which will be discussed. Infected MDMs can seed the
periphery with new infectious virus [20], directly transmit
virus to T cells [27,28], release toxic viral proteins [29-31],
and produce an altered array of cytokines and effector
functions that contribute to HIV pathogenesis [32-35].
Additionally, infected monocyte progenitor cells can har-
bor virus in the bone marrow and seed the periphery with
infected daughter cells. As these cells differentiate in the
marrow and periphery, the levels of HIV-1 transcription
may increase, resulting in the expression of toxic viral pro-
teins and enhanced replication [36] and Alexaki, Shah,
and Wigdahl, unpublished results). These cells can also
cross the blood-brain barrier and deliver virus to the cen-
tral nervous system.
Retroviral gene expression is regulated in a cell type- and
differentiation-dependent manner by the binding of both
host and viral proteins to the long terminal repeat (LTR),
which serves as the viral promoter (reviewed in [37]).
Host transcription factors such as the Sp family, nuclear
factor kappa B (NF-κB) family, activator protein 1 (AP-1)
proteins, nuclear factor of activated T cells (NFAT), and
CCAAT enhancer binding protein (C/EBP) family mem-
bers play key roles in the regulation of retroviral transcrip-
tion by binding sites in the LTR that display different
levels of sequence conservation. Viral proteins such as
HIV Vpr and Tat also bind to the LTR to regulate transcrip-
tion. Many of these host and viral proteins engage in

extensive protein-protein interactions, leading to a com-
plex system of transcriptional regulation. Adding to this
complexity, the genomes of HIV-1, HIV-2, and simian
immunodeficiency virus (SIV) accumulate a significant
spectrum of genetic alterations as the virus replicates.
When present in the LTR, these sequence alterations affect
the ability of host and viral proteins to bind to their cog-
nate binding sites and result in altered transcriptional and
replication potential of the virus [38-46].
Regulation of HIV-1 transcription in cells of the mono-
cyte-macrophage lineage varies considerably with the dif-
ferentiation stage of the cell. Specifically, it has been
observed that cyclin T1 expression in monocytes is con-
trolled by differentiation. Cyclin T1 increases as cells of
the monocyte-macrophage lineage differentiate [47]. This
is important because cyclin T1 is one-half of the positive
transcriptional elongation factor b (P-TEFb) complex nec-
essary for the binding of Tat to TAR for the induction of
HIV-1 transcription. Unstimulated peripheral monocytes
and myeloid progenitor cells support low levels of viral
replication and transcription in response to cellular acti-
vation [27,36,48-54], whereas differentiated MDMs have
increased viral replication but either do not respond to
[45] or downregulate HIV transcription [48,55] in
response to cellular stimulation. During late-stage disease
and AIDS, when CD4
+
T cells have largely been depleted,
HIV-1-infected MDMs represent a greater component of
the total infected cell population, and this pool of virus

contributes significantly to the circulating levels of virus in
vivo [56,57].
Lentiviral LTR Structure
Lentiviral LTRs are comprised of U5, R, and U3 regions.
The U3 region is further divided into the core promoter,
enhancer, and modulatory regions [37]. Lentiviral LTRs,
HIV-1, SIV, and HIV-2, have closely related core promot-
ers (Sp binding sites) and enhancer regions (NF-κB bind-
ing sites) (Fig. 1). These cis-acting elements allow for
efficient replication in a variety of cell types and condi-
tions that result in differential availability and activation
state of transcription factors in the nucleus. However, the
modulatory region is less closely related between lentivi-
ral LTRs and contributes to the ability of the LTR to regu-
late transcription in various cell types and under various
cellular conditions. These concepts are discussed below.
Core promoter and enhancer regions: the interaction of
Sp, NF-
κ
B, and NFAT proteins
Sp factors
The core promoters of HIV-1, HIV-2, and SIV all contain a
TATA box and multiple binding sites for the Sp family of
transcription factors, and their enhancers all contain at
least one binding site for NF-κB. The Sp and NF-κB factor
binding sites in the core promoter play important cell
Retrovirology 2009, 6:118 />Page 3 of 24
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type-specific roles in regulating transcription and replica-
tion. The promoter of HIV-1 contains three binding sites

for Sp factors at -46 to -78 relative to the transcriptional
start site (Fig. 1) [58]. Sp factors also regulate transcription
by binding to positions +271 to +289 [59,60] and -421 to
-451 [61] relative to the transcriptional start site. Sp family
members include Sp1-4, as well as M1 and M2, truncated
Sp3 proteins that result from alternative translational start
sites within the transactivation domain [62-65]. All of the
Sp proteins contain zinc finger DNA binding domains,
and Sp1, 3, and 4 have similar, though not identical, affin-
ities and specificities for GC-rich (GGGGCGGGGC) DNA
[62,66,67]. Sp2 binds to GT-rich sequences (GGTGT-
GGGG) rather than to the GC-rich sequences that consti-
tute the classical Sp binding sites [65]. Sp1 and Sp4 are
transcriptional activators, whereas Sp3 has been classified
as a repressor of HIV-1 transcription. By itself, Sp3 can
weakly activate HIV-1 transcription; however, in the pres-
ence of the strong activator Sp1, it competes for binding
to the LTR and inhibits activation by Sp1 [66,68,69]. In
contrast, M1 and M2 have the Sp3 DNA binding domain
but lack the transactivation domain and are true repres-
sors of transcription in the absence or presence of other Sp
family members [69]. In addition to repressing Sp-medi-
ated transactivation, Sp3 represses LTR activation by the
viral protein Tat [66]. Sp4 is expressed predominantly in
the brain [62,70,71], providing an additional HIV-1 LTR
transactivator to drive replication in this compartment.
Unlike Sp1, Sp4 does not synergistically activate transcrip-
tion in the presence of multiple Sp binding sites [71].
Consequently, the loss of one binding site due to genetic
variation may have less of an effect in the brain than it

would in other tissues, because the loss of function would
not synergistically disrupt binding.
Genetic variation within the Sp sites is likely to play a role
in HIV-1-associated disease progression. The NF-κB-prox-
imal Sp site (site III) is much less conserved during the
course of disease than Sp sites I and II [41] and Kilareski
and Wigdahl, unpublished results). A C-to-T change at
position 5 of Sp site III has been shown to correlate posi-
tively with HIV-1-associated disease progression, both in
the periphery and in the brain [41]. This variant greatly
reduces the affinity of this site for Sp factors, but greatly
increases the response of viral replication to tumor necro-
sis factor α (TNFα) stimulation in peripheral blood
mononuclear cells (Kilareski, Pirrone, and Wigdahl,
unpublished observation). This finding is likely due to a
loss of steric hindrance leading to an increase in NF-κB
binding to its adjacent binding sites (Liu, Banerjee, and
Wigdahl, unpublished observations). In the presence of
Sp4 in the brain, one could speculate that this effect may
be magnified, because Sp4 binding to sites I and II is not
affected by the loss of Sp binding to site III, and the result-
Structure of retroviral LTRsFigure 1
Structure of retroviral LTRs. Retroviral LTRs are divided into the U3, R, and U5 regions, and the U3 region is further
divided into the Modulatory, Enhancer (E) and Promoter regions (top bars). HIV-1, HIV-2, and SIV all contain highly conserved
promoters containing TATA boxes (yellow) and Sp factor binding sites (red) and enhancers (labeled E in light blue bar) contain-
ing NF-κB binding sites (blue). The R region of each contains a trans-acting responsive element (TAR) (orange) that forms an
RNA stem loop structure upon transcription that binds to the viral protein Tat. A negative regulatory element (NRE, pink) was
identified that was subsequently shown to serve as both activator and repressor by binding NFAT proteins (dark blue), AP-1
proteins (purple), and C/EBP factors (green). The modulatory regions of SIVmac and HIV-2 also contain purine box arrays
(PuB, gold) and sites that bind members of the Ets family (teal).

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ing stimulated LTR may have high levels of both Sp and
NF-κB factors bound to their cognate sites.
Sp factors bound to the HIV-1 core promoter cooperate
with the TATA binding protein and TATA binding protein-
associated factors 110 and 55 to drive basal transcription
[72-75]. They can also recruit P-TEFb to promote phos-
phorylation of RNA Pol II [76] and play an important role
in remodeling chromatin to facilitate or inhibit transcrip-
tion [77,78]. Histone deacetylases (HDACs) 1 and 2 are
regulated through phosphorylation by protein kinase
CK2. Sp1 and Sp3 can bind and recruit the phosphor-
ylated HDACs to the LTR to repress LTR activity [79-81].
The repressor activity of Sp1 and Sp3 is regulated by the
expression of CK2 [77].
The three Sp sites in the HIV-1 promoter have different
affinities for Sp factors [39,40,58,82], and the affinity of
Sp for LTR binding sites correlates with replication kinet-
ics; faster viral replication is achieved when a higher affin-
ity Sp binding site is in the NF-κB proximal site [39].
Interestingly, this might, at first glance, seem to contradict
the fact presented above that Sp site III has increased
genetic variation with the 5T variant (a low binding affin-
ity site) correlating with disease progression, given tradi-
tionally low binding affinity correlates with decreased
viral production. However, given that a decreased binding
affinity has been shown to promote higher levels of NF-κB
binding, this variation may actually provide an opportu-
nity for increased replication (Kilareski and Wigdahl,

unpublished observations). This suggests that genetic var-
iations within these sites could have significant effects on
the overall viral replication kinetics [41].
Expression patterns of the different Sp isoforms can mod-
ulate HIV-1 transcription in different cell types. As cells of
the monocyte lineage differentiate, the ratio of Sp1 to Sp3
increases, resulting in increased HIV-1 transcription
(McAllister and Wigdahl, unpublished observations). This
process allows HIV to replicate at low levels, if at all, in cir-
culating monocytes, and to evade the immune system
until the cells are differentiated in peripheral tissues. The
importance of the Sp sites also varies depending on the
differentiation stage of the cell; in unstimulated mono-
cytes, mutation of the Sp sites reduces LTR activity,
whereas in MDMs, transcription of HIV and replication of
SIVmac are abolished when these critical binding sites are
knocked out [83-86].
DNA binding and transactivation activity of Sp factors are
regulated both positively and negatively by phosphoryla-
tion and other post-translational modifications (reviewed
in [87,88] and Fig. 2). Phosphorylation at Sp1 Ser131 by
DNA-dependent protein kinase increases the affinity of
the protein for DNA and also increases the ability of the
protein to cooperate with the viral protein Tat to transac-
tivate the LTR [89-92]. In contrast, O-linked N-acetylglu-
cosaminylation (O-GLcNAc) of Sp1 inhibits HIV-1
replication [93]. Therefore, modulating O-GLcNAc of
transcription factors may play a role in regulation of HIV-
1 latency and activation, and may link glucose metabo-
lism to HIV-1 replication.

NF-
κ
B
NF-κB proteins have been shown to be one of the main
modulators of the HIV-1 LTR in all cell types and a poten-
tial pathway for anti-HIV-1 therapies [94]. NF-κB proteins
bind the enhancer at two sites located at nucleotide posi-
tions -81 to -91 and -95 to -104 relative to the transcrip-
tional start site [95-97]. NF-κB is composed of
heterodimers of five c-rel protein family members: p65/
RelA, NF-κB1/p50, c-Rel, RelB, and NF-κB2/p52. Func-
tional NF-κB in T cells is predominantly composed of p65
or c-Rel bound to p50 or p52, whereas in MDMs, Rel B
replaces p65 [97-100]. In T cells and immature mono-
cytes, NF-κB shuttles between the cytoplasm and the
nucleus in response to cellular stimuli. In the cytoplasm,
NF-κB is bound to inhibitor (IκB) proteins [101]. As a
result of specific stimuli, IκB is phosphorylated and
released from NF-κB; after release from the inhibitory
complex, NF-κB translocates to the nucleus where it acti-
vates many host and viral genes through the initial recruit-
ment of P-TEFb (Fig. 3) [101-103]. Interestingly, one of
the IκB's, IκBα has been shown to play a role in shuttling
of NF-κB from the nucleus and cytosol and in the binding
NF-κB in the nucleus of T cells, potentially contributing to
the lower activation levels of the HIV-1 LTR and possibly
promoting viral latency [104]. However, this mechanism
has not been explored in cells of the monocyte-macro-
phage lineage. NF-κB can also function as a repressor of
transcription through the recruitment of HDAC1 (Fig. 3)

[78,105].
NF-κB DNA binding activity first occurs in monocytes as
they progress from promonocytes to monocytes; however,
in mature monocytes and MDMs, NF-κB is constitutively
active in the nucleus, and its DNA binding activity is not
increased further in response to cellular activation or dif-
ferentiation [106]. This constitutive pool of NF-κB allows
a low level of basal HIV transcription in the absence of cel-
lular stimuli. Binding of NF-κB to the enhancer of the
HIV-1 LTR plays a critical role in the response of the LTR
to cellular stimuli in both T cells and maturing monocytes
[36,94,97,106-109]. Deletion or mutation of the NF-κB
sites abolishes LTR activity [97,109-112] and results in
reduced production of infectious virus [98]. Activation of
monocytes by LPS, IL-6, or TNF-α (Fig. 3) results in
enhanced HIV replication, a process that correlates with
activation of NF-κB [27,49-51,113]. LPS activation of
monocytes leads to the induction of the NF-κB pathway
Retrovirology 2009, 6:118 />Page 5 of 24
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through TNF-α [27,50]. In contrast, in differentiated pri-
mary MDMs, stimulation by LPS results in the downregu-
lation of LTR activity and viral replication [48]. This
activity was not affected by mutation of the NF-κB sites,
but did map to the enhancer element (position -156 to -
121); thus, this effect may involve NFAT proteins (see
below) [48]. While this may seem counter-intuitive, one
might speculate that stimulation of cells through the NF-
κB pathway would enhance LTR activity and viral replica-
tion, it should be noted that LPS stimulation of differenti-

ated macrophages could also induce transcription factors
that negatively regulate the LTR, however this has not
been explored. This would be very interesting as this
might provide another reason for macrophages serving as
a latent reservoir for HIV-1. In addition to activating tran-
scription by binding the enhancer region, NF-κB activates
transcription by binding to sites -1 to +9 and +31 to +40
relative to the transcriptional start site [114,115].
The NF-κB site(s) located immediately upstream of the Sp
sites in the enhancer in HIV and SIV result in Sp-NF-κB
protein-protein interactions that further modulate the
LTR activity. Sp1 and NF-κB proteins bind the LTR coop-
eratively and activate transcription synergistically in
response to cellular stimulation [66,82,109]. This activa-
tion is mediated by the binding of the DNA-binding
domain of p65 to the DNA-binding domain of Sp1 [108]
(Fig. 4). Sp3 and Sp4 are unable to activate transcription
cooperatively with NF-κB [66]. In the absence of func-
tional Sp sites (or in the presence of genetic alterations
that inactivate the Sp binding sites), binding of NF-κB to
the enhancer can restore replication of the virus in T cells
[116-118], perhaps by recruiting Sp to the variant sites.
NFAT (AP-3)
NFAT proteins are part of a family of Rel-related transcrip-
tion factors that become active early after T cell activation
and are constitutively in monocytes. NFAT exists as several
Important Sp transcription factor signaling in monocyte-macropahgesFigure 2
Important Sp transcription factor signaling in monocyte-macropahges. (a) Activation of HIV transcription by the
interaction of viral protein Tat with DNA-dependent protein kinase (DNA-PK) results in the subsequent phosphorylation at
Ser131 of Sp1. Phosphorylated Sp1 results in increased transcription of proviral DNA, resulting in an increase in Tat produc-

tion, perpetuating the cycle. (b) Inhibition of HIV transcription involves O-linked N-acetylglucosamine (O-GlcNAc) transferase
(OGT) catalyzing the addition of O-GlcNAC to Sp proteins which blocks their interaction with their binding sites on the LTR,
resulting in an inhibition/reduction in HIV transcription.
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isoforms (NFAT1, NFAT2/NFATc, and NFAT3-5) that acti-
vate a variety of genes in immune and non-immune cell
populations [119-121]. Like NF-κB, NFAT contains a
DNA-binding domain that is homologous to rel and shut-
tles between the cytoplasm and nucleus in response to cel-
lular stimuli [122,123]. In the cytoplasm, NFAT is
dephosphorylated and translocates to the nucleus where it
activates transcription of many genes [124-126] (Fig. 4).
NFAT can bind DNA as a high affinity dimer or as a lower
affinity monomer [127-129]. NFAT proteins frequently
cooperate with other transcription factor families when
bound to adjacent sites within a promoter.
An NFAT binding site was identified in the HIV-1 LTR at
positions -216 and -254, with a footprint extending from
-253 to -215 relative to the transcriptional start site
[122,130]. Although this site can bind NFAT in vitro, this
site was later shown not to be necessary for NFAT-medi-
ated activation of the HIV-1 LTR [131,132]. Instead, NFAT
binds the NF-κB binding sites in the enhancer in response
to cellular activation in T cells and constitutively in mono-
cytes [110,112,127,130,133]. NFAT activation of genes
from κB-like sequences has been documented with a
number of host and viral promoters [134,135] (Fig. 4). In
addition to binding to the enhancer, NFAT binding at
positions +169 to +181 has been reported to activate tran-

scription [59,60,136].
NFAT proteins activate HIV-1 transcription and replica-
tion in a variety of cell types. Whereas NFAT1 and NFAT2/
NFATc are responsible for the activation of HIV in T cells
[110,133,137] reviewed in [138,139]), NFAT5, the most
evolutionarily divergent NFAT member, regulates HIV
replication in monocyte-MDMs [130]. Terminally differ-
entiated MDMs constitutively express high levels of
NFAT5, which is able to bind and activate the enhancer of
Important NF-κB transcription factor signaling in monocyte-macrophagesFigure 3
Important NF-κB transcription factor signaling in monocyte-macrophages. (a) Activation of HIV transcription:
Translocation of NF-κB from the cytoplasm to the nucleus is controlled by association of IκB with the NF-κB hetero-/homo-
dimer. Once IκB is phosphorylated, it relesases NF-κB which then translocates to the nucleus where it can bind the LTR and
induce HIV transcription. (b) Inhibition of HIV transcription: In T cells, IκBα has been shown to contribute to lower levels of
LTR transcription and potentially contribute to latency. It is postulated that a similar mechanism of action could be in place for
cells of the monocyte-macrophage lineage. In addition, NF-κB's association with the histone deacetylase inhibitor HDAC1
results in constriction of the chromatin so that RNA polymerase does not have access to its target DNA.
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HIV-1 subtypes B, C, and E, HIV-2, and SIV from multiple
primate species [130]. Targeting NFAT5 with siRNAs in
primary MDMs modestly reduces viral replication [130];
however, NFAT derived from MDM nuclear extract was
unable to compete with NF-κB for binding to the HIV
enhancer in vitro [98]. This finding suggests that in vivo,
although constitutively expressed NFAT is able to bind the
LTR, it is unable to do so in the presence of high levels of
NF-κB.
Modulatory region
As its name implies, the modulatory region of the LTR

functions to regulate transcription that is driven by the
core and/or enhancer regions. A wide array of host and
viral proteins bind the modulatory region of the LTR to
either enhance or repress transcription [45,46,140]. In
HIV-1, the loss of both the Sp and NF-κB sites effectively
inactivates the LTR. In contrast, the modulatory region of
SIVmac and HIV-2 have functional elements that are not
present in HIV-1 that can compensate, at least in part, for
the loss of the Sp and NF-κB sites [85]. Also, unlike the
HIV-1 LTR, the 5' 364 bp of the 517 bp-long U3 region is
dispensable for SIV replication [141-143]. Early reports
investigating the role of the HIV-1 modulatory region
identified bases -423 to -167 as a negative regulatory ele-
ment (NRE) that repressed LTR activity [144]. Since then,
this region has been shown to activate as well as to repress
transcription (for review see [140]).
Basic leucine zipper transactivator proteins
C/EBPs, activating transcription factor/cyclic AMP
response element binding (ATF/CREB) proteins, and AP-
1 factors are members of a large family of basic leucine
zipper (bZIP) proteins that play important roles in the
Important C/EBP transcription factor signaling in monocyte-macrophagesFigure 4
Important C/EBP transcription factor signaling in monocyte-macrophages: (a) Activation of HIV transcription: C/
EBP, located in the cytoplasm of the cell, can become phosphorylated by the MAP kinase, PKA, or cdk9 through a variety of
pathways. Once phosphorylated, C/EBP is translocated into the nucleus where it can transactivate the LTR. In addition, C/EBP
associates with histone acetyl transferases such as p300, which when bound to the LTR, make the chromosome accessible for
RNA polymerases to bind and transcribe the integrated proviral DNA. Finally, association of C/EBP with APOBEC3G allows
for better reverse transcription in the cytoplasm. (b) Inhibition of HIV transcription: The binding of IFNβ to its receptor begins
a JAK/STAT signaling cascade that results in increased production of C/EBP3 (LIP). C/EBP3, which does not contain the trans-
activation domain of full-length C/EBPs, does not interact with histone acetyl transferases and when bound to the LTR, blocks

the binding of full-length C/EBPs, thereby leading to a repression of LTR activity.
Retrovirology 2009, 6:118 />Page 8 of 24
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regulation of retroviral transcription [145-147]. Dimeri-
zation of the bZIP family members occurs in the C-termi-
nal α-helical leucine zipper domain and is necessary for
binding to DNA (reviewed in [148,149]). C/EBP, AP-1,
and ATF/CREB proteins each have unique binding sites in
the modulatory region of the HIV-1 LTR; however, het-
erodimerization between C/EBP and ATF/CREB or AP-1
family members has been shown to result in binding to
sequences that are different from the consensus sequence
for either family of factors [146,150-155]. These
sequences are often composed of half of the recognition
sequence for each protein in the heterodimer [146,150].
C/EBP
The HIV-1 LTR contains three C/EBP binding sites
upstream of the transcriptional start site [156,157] and
one binding site downstream of the transcriptional start
site, at the 3'-most end of U5 (Liu and Wigdahl, unpub-
lished observations). C/EBPs play a critical role in HIV-1
replication. It has been shown that at least one upstream
C/EBP binding site and the presence of C/EBP proteins are
necessary for replication in cells of the monocyte-macro-
phage lineage [157-161]. The two C/EBP binding sites
located in the U3 region of the LTR have differing affini-
ties for C/EBP factors, with the upstream site (site II), hav-
ing a much higher relative affinity than the downstream
site (site I) [43]. In addition to activating HIV-1 transcrip-
tion through direct binding to the LTR, C/EBP factors may

inhibit the host cellular antiviral protein APOBEC3G (Fig.
5), allowing more efficient reverse transcription to occur
in the cytoplasm [162].
The C/EBP family of transcription factors consists of six
members, including C/EBP α, β, γ, δ, ε, and ζ [163-169].
C/EBPβ itself has three isoforms that result from the use
of internal start codons within a single mRNA [170,171].
C/EBP-1, the full-length isoform, and C/EBP-2, an iso-
form that lacks the N-terminal 23 amino acids, both con-
tain three transcriptional activation domains and
function as activators of HIV-1 transcription. C/EBP-3,
which lacks the N-terminal 198 amino acids that include
the activation domains, serves as a repressor of HIV-1
transcription, because it retains the C-terminal DNA-
binding domain and competes for binding with the acti-
vator isoforms of C/EBP.
C/EBP isoform expression depends on the differentiation
and activation state of cells in the monocyte-macrophage
lineage. C/EBPα levels are high early in monocyte differ-
entiation and then decrease as cells mature, whereas C/
EBPβ and C/EBPδ levels are low early in development and
increase as cells mature [172,173]. C/EBP isoform expres-
sion is also regulated by extracellular stimuli. C/EBPβ
expression increases upon cellular activation, whereas
expression of the other C/EBP isoforms remains constant
[172,174]. Exposure of macrophages to interleukin-1 (IL-
1), tumor necrosis factor α (TNFα), or interferon-γ, all of
which have been shown to be present at elevated levels
during the course of HIV-1 infection, has been shown to
induce a reduction in C/EBPα mRNA levels while the lev-

els of C/EBPβ and C/EBPδ expression increase [174]. This
results in C/EBPβ and C/EBPδ playing a key role in the
regulation of HIV-1 transcription as disease progresses
and inflammatory cytokine levels increase (Fig. 4).
An additional level of regulation of C/EBPβ activity
resides in two regulatory domains that lie between the
activation domains and the DNA binding domain. These
domains inhibit C/EBP activity, until phosphorylation
results in an increase in DNA binding affinity and tran-
scriptional activation activity [175,176]. Several signaling
cascades regulate the phosphorylation state of C/EBP.
Phosphorylation of threonine 235 by a ras-dependent
mitogen-activated protein kinase increases transcriptional
activation [177]; phosphorylation of serine 288 by cAMP-
dependent protein kinase A results in nuclear transloca-
tion and subsequent transactivation [178]; and cyclin-
dependent kinase 9 (cdk9) phosphorylates C/EBPβ and
leads to an increase in HIV-1 gene expression [179] (Fig.
4).
C/EBPs interact with many nuclear proteins to activate
transcription. In addition to binding other bZIP proteins,
C/EBP recruits chromatin remodeling complexes such as
SWI/SNF[180], cAMP response element-binding protein/
p300 [181,182], and p300/CREB-binding protein-associ-
ated factor [183] to the HIV-1 LTR. These proteins
remodel the chromatin structure and increase transcrip-
tion of the HIV-1 genome. C/EBP increases the phospho-
rylation of p300, which in turn alters its nuclear
localization and increases its activity [184]. C/EBP can
also act synergistically with Sp proteins to activate tran-

scription of the HIV-1 LTR [185].
The importance of C/EBP factors in the regulation of HIV-
1 gene expression is underscored by the discovery that a
6G configuration (a T-to-G change at nucleotide position
6) in C/EBP site I increases C/EBP binding, increases LTR
activity, and is preferentially encountered in proviral LTRs
derived from the brain of HIV-1-infected patients
[42,186]. C/EBP site II was also found to be preferentially
conserved in the consensus subtype B configuration or to
contain a 6G variation of this site, which are both high
affinity sites for C/EBP factors in LTRs present in proviral
DNA in cells located in the mid-frontal gyrus of the brain
of infected individuals. A high rate of viral replication
occurs in this region of the brain. Interestingly, the pres-
ence of the 6G configuration of this binding site also cor-
relates with the presence of HIV-1-associated dementia
[42,44]. In contrast, the presence of a 4C C/EBP site II,
Retrovirology 2009, 6:118 />Page 9 of 24
(page number not for citation purposes)
which is a low-affinity C/EBP site, has been found prefer-
entially in the cerebellum, a region of low viral replication
[44]. This observation suggests that high affinity for C/
EBP factors may contribute to the maintenance and/or
pathogenesis of HIV-1 in the central nervous system,
whereas low affinity sites such as 4C may contribute to
lower levels of transcription required to maintain a latent
reservoir of provirus. We have also identified a 3T config-
uration (a C-to-T change at position 3) of C/EBP site I that
exhibits a low affinity for C/EBP within LTRs in the
peripheral blood and brain and has also been shown to

correlate with both late stage HIV disease and HIV-1-asso-
ciated dementia [43], respectively.
Regulation of HIV-1 transcription in circulating monocytesFigure 5
Regulation of HIV-1 transcription in circulating monocytes. Transcription of HIV-1 in circulating monocytes is depend-
ent on the ratio of activator to repressor isoforms of transcription factors, the phosphorylation state of transcription factors,
and the inducible translocation of NF-κB and NFAT factors from the cytoplasm. NF-κB can be induced to translocate to the
nucleus by TNFα-mediated phosphorylation of IκB. NFAT is dephosphorylated in the cytoplasm by calcineurin, which acts in
response to calcium levels within the cell. Once it is dephosphorylated, it translocates to the nucleus where it activates tran-
scription by constitutively binding the NF-κB site in the enhancer. Phosphorylation plays a critical role in regulating the activity
of C/EBP factors in monocytes. Phosphorylation of C/EBPα by ras-dependent mitogen-activated protein (MAP) kinase, signaled
by IL-6 or by cAMP-dependent protein kinase A, results in its nuclear translocation and subsequent transactivation of the LTR.
Cyclin-dependent kinase (cdk) 9 specifically phosphorylates C/EBPβ, which then translocates into the nucleus, binds to the
LTR, and leads to an increase in HIV-1 gene expression. Once in the nucleus, C/EBP factors then regulate the activity of AP-1
factors. Relatively high levels of C/EBPα dimerize with AP-1 factors to form potent activators of transcription. Lower levels of
C/EBPβ balance this activation by binding AP-1 leading to a loss in DNA binding affinity. Sp1 and Sp3 are constitutively
expressed in the nucleus. In the presence of Sp1, which is a strong activator, Sp3 competes for binding to the LTR and inhibits
activation by Sp1.
Retrovirology 2009, 6:118 />Page 10 of 24
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ATF/CREB
ATF/CREB binds the HIV-1 LTR at a site immediately
upstream of the C/EBP binding site I [38,187] and at two
sites downstream of the transcriptional start site (sites
+160 to +167 and +92 to +102) to regulate the LTR
[59,60,188,189]. ATF/CREB and C/EBP factors can bind
their adjacent upstream sites individually as homodimers,
or C/EBP and ATF/CREB can heterodimerize with each
other to regulate HIV-1 expression. This heterodimeriza-
tion results in the recognition of a site composed of the 3'
half of the ATF/CREB site and the 5' half of the C/EBP site

[146]. As a result, in the presence of genetic variation that
results in a low affinity C/EBP site, ATF/CREB is able to
recruit C/EBP factors to the site and vice versa [146]. In
addition to activating transcription, ATF/CREB can inhibit
transcription by binding to Swi6, a component of the
remodeling complex SWI/SNF, to promote the formation
of heterochromatin [190].
AP-1 (Fos/Jun)
AP-1 proteins exist as homodimers of Jun family members
(c-Jun, JunB, and JunD) or as heterodimers of Jun and Fos
family members (c-Fos, FosB, Fra-1, and Fra-2) (reviewed
in [191]). They bind a palindromic DNA sequence known
as the TPA-responsive elements (TRE) at positions -306 to
-285 and -242 to -222 of the LTR [59] as well as at posi-
tions +95 and +160, downstream of the transcriptional
start site [59,60,188,189]. The sequence of these sites has
been shown to evolve in a manner that facilitates efficient
cell type-specific binding of AP-1 [59,192]. AP-1 acts as
either an activator or repressor of transcription, depend-
ing on the components of the dimer [191,193]. Once
bound to the promoter, cFos/cJun heterodimers can
recruit the SWI/SNF chromatin remodeling complex to
activate transcription, whereas homodimers or het-
erodimers consisting of other family members lack this
ability [194].
AP-1 mRNA is typically absent in quiescent cells; however,
it is significantly up-regulated upon cellular stimulation
[195]. Jun levels increase during monocytic maturation
and become constitutively expressed in MDMs [196-200].
Despite being expressed, AP-1 in MDMs of some tissues,

such as the lung, lacks the ability to bind DNA because of
the lack of expression of Ref-1, a protein that modulates
the oxidation state of Fos [201,202]. In addition to being
regulated by oxidation [201-203], AP-1 protein activity is
further controlled post-transcriptionally by sumoylation,
which inhibits protein activity [204,205], and by phos-
phorylation, which increases activity in response to cellu-
lar stimulation [206].
In addition to directly regulating HIV-1 gene expression,
AP-1 proteins can modulate the activity of other transcrip-
tion factors. C/EBPβ dimerization with c-Fos or c-Jun
results in C/EBP being unable to bind DNA thus a reduc-
tion in C/EBP-mediated transactivation [153,154]. In
contrast, C/EBPα dimerization with c-Jun or c-Fos forms a
potent activator of transcription [207]. In response to
mitogen or cytokine stimulation, the mitogen-activated
protein kinases ERK1/ERK2 phosphorylate AP-1
(reviewed in [208] and [209]). This phosphorylation pro-
motes the interaction of AP-1 with NF-κB and the
enhancer element, which leads to the synergistic activa-
tion of the LTR [210-213]. This cascade of events is one
mechanism by which HIV emerges from latency
[210,214].
Tat
Tat is a virus-encoded transcriptional transactivator that
binds to the RNA secondary structure encoded by the
transactivation region (TAR) in the repeat segment of the
LTR (+1 to +59) [215,216]. Once bound to the elongating
transcript, Tat helps assemble the pre-initiation complex
and recruits cdk9 to promote phosphorylation of RNA Pol

II [217,218] and P-TEFb to increase processivity of RNA
Pol II [219-223]. Interestingly, mechanistic studies of this
complex suggest that one of the functions of Tat is to
increase the duration of P-TEFb occupancy at the HIV-1
LTR [224]. Tat also significantly remodels chromatin by
recruiting the histone acyltransferases Tip60 [225,226],
human Nucleosome Assembly Protein-1 (hNAP-1) [227],
p300/cAMP response element-binding protein [228,229],
and p/CAF [230], as well as the chromatin-remodeling
complex SWI/SNF [231]. Tat activity is limited in mono-
cytes due to the lack of sufficient levels of cyclin T1, a com-
ponent of P-TEFb [54]. Differentiation into macrophages
increases Cyclin T1 expression and results in strong Tat
activity [54].
Tat regulates the activity of many other transcription fac-
tors through direct protein-protein interactions and the
modulation of kinase activities. Tat promotes the phos-
phorylation of Sp1, which in turn increases binding of Sp
to the LTR [92]. Conversely, Sp is also necessary to recruit
Tat to the LTR [76], and deletion or mutation of the Sp
binding sites in the promoter abolishes Tat activity
[232,233]. It is currently unclear whether direct interac-
tion occurs between Sp factors and Tat [234-236]. In addi-
tion to regulating Sp1 activity, Tat increases the
cooperation between NFAT and AP-1 proteins without
altering independent binding of these transcription fac-
tors to DNA [137,237]. It also promotes the interaction of
NF-κB and AP-1 factors to synergistically activate tran-
scription [238-240].
Vpr

Vpr is another virus-encoded protein that plays a direct
role in the regulation of HIV-1 transcription [241-243].
Vpr is found in the viral particle and plays an important
Retrovirology 2009, 6:118 />Page 11 of 24
(page number not for citation purposes)
role in early transcriptional activation of the LTR before
Tat can be expressed [244-248]. Its importance is high-
lighted by a recent study that describes alterations in Vpr
that provide a significant reduction in Vpr nuclear import
and virion incorporation uniquely in a long term non-
progressor patient [249]. Vpr also causes cell cycle arrest in
the G2 phase, the phase of the cell cycle when the LTR is
most active, which results in apoptosis. [250]. It is neces-
sary for viral replication in cells of the monocyte-macro-
phage lineage [251-256]. Interestingly, Vpr has been
shown to interact with the nuclear form of uracil DNA gly-
cosylase (UNG2), a cellular DNA repair enzyme, which
helps incorporate this protein into virus particles leading
to a decrease in viral mutation rate. Specifically, the lack
of UNG in virions during virus replication in primary
monocyte-derived macrophages further increases virus
mutant frequencies by 18-fold compared with the 4-fold
increase measured in actively dividing cells [257]. In addi-
tion, Vpr has been shown to concentrate at the nuclear
envelope (NE) shortly after infection (4-6 hrs) as part of
the pre-integration complex (PIC), supporting an interac-
tion between Vpr and components of the nuclear pore
complex [258-261], including the nucleoporin hCG1
[262]. Single-point Vpr mutants within the first α-helix of
the protein such as Vpr-L23F and Vpr-K27M fail to associ-

ate with hCG1, but are still able to interact with other
known relevant host partners of Vpr. In primary human
monocyte-derived macrophages, these mutants fail to
localize at the NE resulting in a diffuse nucleocytoplasmic
distribution, impaired the Vpr-mediated G2-arrest of the
cell cycle, and subsequently induced cell death. These
observations were obtained in primary macrophages from
some but not all donors indicating that the targeting of
Vpr to the nuclear pore complex may constitute an early
step toward Vpr-induced G2-arrest and subsequent apop-
tosis. These results also suggest that Vpr targeting to the
nuclear pore complex is not absolutely required, but can
enhance HIV-1 replication in macrophages [263]. Extra-
cellular Vpr is found in the plasma and the CSF [254,264]
and can enter monocytes and macrophages and behave as
if the protein was endogenously expressed [265-267]. Vpr
binds the LTR in a sequence-specific manner to activate
transcription directly [45,46] and also interacts with Sp1
[268], TFIIB [269,270], NF-κB [271], C/EBP [272], and
Tat [244,273] to enhance transcription of the HIV-1
genome. Vpr activates the DNA binding activity of AP-1 by
promoting the phosphorylation of cFos and cJun in
monocytes and macrophages [267]. It also promotes the
translocation of NF-κB p50/p65 to the nucleus by pro-
moting the phosphorylation of IκB [267], which allows
an NF-κB- and AP-1-mediated increase in LTR activity.
C/EBP and Vpr interact at the HIV-1 LTR in two ways. Vpr
has been shown to increase C/EBPβ DNA binding activity
[272]. It has also been shown that Vpr has a high affinity
for LTR C/EBP binding site I variants that exhibit a

decreased affinity of the site for C/EBP. The presence of
these LTR variants correlates with late-stage HIV-associ-
ated disease [45,46]. Thus, as HIV-1-associated disease
progresses, viral variants containing this type of LTR C/
EBP site I may become more prevalent and function to
facilitate a transition from C/EBP-mediated LTR activation
to Vpr-mediated transactivation from that site. Alterna-
tively, Vpr and C/EBP may form a complex at that site
(Burdo and Wigdahl, unpublished observations). In addi-
tion to interacting with cellular proteins, Vpr interacts
with Tat and activates transcription in an additive manner
[244,274].
Methylation
HIV proviral DNA that has integrated into the host
genome also becomes subject to host factors that regulate
chromatin organization and gene transcription. These
mechanisms include histone modification, RNA interfer-
ence/silencing, and DNA methylation. The mechanisms
play a role in the control of gene expression, viral activa-
tion, and/or latency. DNA methylation of CpG islands
within the HIV-1 LTR is one process that results in the
downregulation/silencing of the integrated proviral
genome [275-278]. This form of transcriptional silencing
occurs by specific methyltransferases that are directed to
the target DNA by methylation of lysine 9 of histone H3
through histone methyltransferases [279]. In cells of the
monocyte-macrophage lineage, methylation of the LTR
has been found to result in the transcriptional silencing of
the promoter which contributes to limited access of tran-
scription factors to the target DNA [280]. In addition, in

the CD4
+
T cell line ACH-2, the transcriptional silencing
brought about by DNA methylation of the LTR can be
reversed through TNF-α treatment of the cells which leads
to demethylation of the 5' LTR and the induction of viral
gene expression [281] showing that although this modifi-
cation is inheritable, it is not permanent. The reduction of
LTR expression is possibly explained by the binding of
methyl-CpG-binding protein 1 complex and methyl-
CpG-binding protein 2 to methylated Sp1 transcription
factor binding sites, thereby inhibiting the binding of Sp1
transcription factors [282,283]. In addition, the transcrip-
tion factors USF and NF-κB lose affinity for their methyl-
ated LTR transcription factor binding sites as well [284].
Unfortunately, to date all of these studies have been per-
formed in T cell lines and primary T cells, but not in cells
of the monocyte-macrophage lineage.
Cytokines
Cytokines play a critical role in the pathogenesis of HIV-
1. IL-6, TNFα, IL-1β, and other proinflammatory cytokine
levels are elevated in the blood, bone marrow, and cere-
brospinal fluid of HIV-infected patients [285,286]. IL-6
and TNF-α are induced early after HIV monocytic infec-
Retrovirology 2009, 6:118 />Page 12 of 24
(page number not for citation purposes)
tion, followed by their continued increased expression
[52,53,287]. IL-6 is a potent activator of C/EBP, and expo-
sure of monocytes to IL-6 results in increased HIV-1 repli-
cation. The increase in C/EBP activity then forms a

positive feedback loop for IL-6 expression, because C/
EBPβ binds to and activates the IL-6 promoter [288]. C/
EBPs can also activate the genes encoding other proin-
flammatory cytokines such as IL-1β [289] and TNFα
[290,291]. TNFα is one of the most potent activators of
NF-κB activity known. It acts by causing a signaling cas-
cade that activates the IκB kinase complex, which then
phosphorylates IκB, releasing NF-κB. The free NF-κB
translocates to the nucleus and induces the activation of
the HIV-1 LTR (Fig. 3 and 4).
In addition to being regulated by cytokines, chemokines
contribute to HIV-1 infection and pathogenesis. The HIV-
1 Nef protein induces HIV-infected macrophages to
secrete at least two chemokines, MIP1α and MIP1β, which
recruit and activate resting CD4+ T lymphocytes [292].
These T cells can then become infected and produce high
levels of virus.
Summary of important monocytic regulatory pathways
regulating the HIV-1 LTR
Regulation of HIV-1 transcription in cells of the mono-
cyte-macrophage lineage varies considerably with the
stage of cellular differentiation as well as in comparison to
activated T cells. Specifically, it has been observed that cyc-
lin T1 expression in monocytes is controlled by differenti-
ation. Cyclin T1 increases as cells of the monocyte-
macrophage lineage differentiate [47]. Unstimulated
peripheral blood monocytes and myeloid progenitor cells
support low levels of viral replication and activate tran-
scription in response to cellular activation like T cells
[27,36,48-54] whereas differentiated MDMs have

increased viral replication but either do not respond to
[45] or downregulate HIV transcription [48,55] in
response to cellular stimulation. As cells of the monocyte
lineage differentiate, the ratio of Sp1 to Sp3 increases,
resulting in an increase in HIV-1 transcription (McAllister
and Wigdahl, unpublished observations). This process
may result in low level HIV replication, or viral genomic
silence, in circulating monocytes, and evasion of the host
immune system until the cells are differentiated in periph-
eral tissues. The importance of the Sp sites also varies
depending on the differentiation stage of the cell; in
unstimulated monocytes, mutation of the Sp sites reduces
LTR activity, whereas in MDMs, transcription of HIV and
replication of SIVmac are abolished when these critical
binding sites are knocked out [83-86]. NF-κB regulation
of the LTR is also unique in MDMs. In MDMs, NF-κB is
composed of Rel B bound to p50 or p52, whereas NF-κB
in T cells is predominantly composed of p65 or c-Rel
bound to p50 or p52 [97-100]. NF-κB DNA binding activ-
ity first occurs in monocytes as they progress from prom-
onocytes to monocytes; however, in mature monocytes
and MDMs, NF-κB is constitutively active in the nucleus,
and its DNA binding activity is not increased further in
response to cellular activation or differentiation [106].
Stimulation of T cells and monocytes by LPS results in
enhanced HIV replication, a process that correlates with
activation of NF-κB [27,49-51,113]. In differentiated pri-
mary MDMs, stimulation by LPS results, however, in the
downregulation of LTR activity and viral replication [48].
NFAT, C/EBP, Jun and AP-1 transcription factor regulation

of LTR activity also have distinct differences in monocyte-
macrophages compared to T cells. NFAT binds the NF-κB
binding sites in the enhancer in response to cellular acti-
vation in T cells but binds constitutively in monocytes
[110,112,127,130,133]. Also, NFAT5, the most evolution-
arily divergent NFAT member, regulates HIV replication in
monocyte-MDMs [130] but has not been shown to do this
in T cells. With regard to C/EBP, it has been shown that at
least one upstream C/EBP binding site and the presence of
C/EBP proteins are necessary for replication in cells of the
monocyte-macrophage lineage but not in T cells [157-
161]. Jun levels increase during monocytic maturation
and become constitutively expressed in MDMs [196-200].
Despite being expressed, AP-1 in MDMs of some tissues,
such as the lung, lacks the ability to bind DNA because of
the lack of expression of Ref-1, a protein that modulates
the oxidation state of Fos [201,202].
The viral proteins Tat and Vpr have also been shown to
have unique properties with regard to HIV-1 LTR activa-
tion in cells of the monocyte-macrophage lineage. Tat
activity has been shown to be limited in monocytes due to
the lack of sufficient levels of cyclin T1, a component of P-
TEFb [54]. Differentiation into macrophages increases
Cyclin T1 expression and results in strong Tat activity [54].
Vpr has been shown to be necessary for viral replication in
cells of the monocyte-macrophage lineage but not in T
cells [251-256]. Vpr has also been shown to specifically
play a role in viral mutation rates in cells of the monocyte-
macrophage lineage. Specifically, the lack of UNG in viri-
ons due to lack of Vpr binding to UNG during viral pack-

aging led to increased virus mutant frequencies as
indicated previously (18-fold increase compared to a 4-
fold increase) [257]. In addition, genetic variation in Vpr
has been shown in primary human monocyte-derived
macrophages to fail in Vpr localization at the NE resulting
in a diffuse nucleocytoplasmic distribution, impairing the
Vpr-mediated G2-arrest of the cell cycle and the subse-
quent cell death induction, in some but not all donors
[263].
Retrovirology 2009, 6:118 />Page 13 of 24
(page number not for citation purposes)
Conclusions
Regulation of HIV-1 transcription in cells of the mono-
cyte-macrophage lineage is a complex process involving
the interaction of numerous factors that are expressed in a
differentiation-dependent manner and whose activity is
regulated by both cellular differentiation and extracellular
signaling pathways. Although monocytes can be infected,
this process is hindered at multiple steps in the viral life-
cycle, including transcription. The mechanism behind the
block to replication in monocytes has yet to be fully char-
acterized, but it is clear that many factors make contribu-
tions. Monocytes express relatively low levels of the HIV
co-receptor CCR5 [293,294] and recently, it has been
shown that viral entry is impaired in circulating mono-
cytes [295]. Reverse transcription and integration are also
impaired [295,296]. At the transcriptional level, LTR activ-
ity is regulated by the ratio of activator to repressor iso-
forms of transcription factors, the phosphorylation state
of transcription factors, the inducible translocation of NF-

κB and NFAT factors from the cytoplasm, and the availa-
bility of viral transactivator proteins and their host co-fac-
tors (Fig. 4). Members of the AP-1 transcription factor
family and relatively equal levels of nuclear Sp1 to Sp3
facilitate a modest level of basal transcription, whereas
NF-κB and NFAT proteins remain sequestered in the cyto-
plasm in the early stages of monocytic differentiation. The
presence of Tat has little effect on transcription in mono-
cytes, as cyclin T1 expression is undetectable and other
factors required for Tat activation are absent [54]. This
lack of Tat activity contributes to replication block
observed in unstimulated circulating monocytes.
Although circulating monocytes exhibit low levels of viral
replication, replication increases in response to cytokine
stimulation. During periods of inflammation caused by
HIV-1 infection, co-pathogens, or opportunistic infec-
tions, levels of circulating cytokines such as IL-6 and TNFα
increase and stimulate HIV-1 replication in monocytic
cells. IL-6 increases the activity of C/EBP factors; these fac-
tors then activate the LTR and form a positive feedback
loop by activating the promoters of cytokines, including
TNFα and IL-6. In response to TNFα, NF-κB and NFAT5
translocate to the nucleus, and AP-1 DNA binding activity
is stimulated to activate transcription as a result of
changes in protein phosphorylation (Fig. 6). NFAT and
NF-κB interact at the enhancer to activate transcription
synergistically, whereas ATF/CREB, AP-1, and C/EBPα, β,
and δ form homo- and heterodimers to regulate LTR activ-
ity. Vpr binds to the LTR directly and through interactions
with other factors associated with the transcriptional com-

plex in conjunction with AP-1, NF-κB, and C/EBP to acti-
vate transcription.
As monocytes differentiate into macrophages, the permis-
siveness to viral replication increases dramatically,
although MDMs lose the ability to further increase viral
replication in response to extracellular stimuli. Cofactors
necessary for Tat transactivation of the LTR are expressed,
allowing a much greater level of HIV-1 transcription than
is possible in monocytes. NF-κB, AP-1, and NFAT proteins
are constitutively localized in the nucleus, and the activa-
tor Sp1 expression predominates over the repressor Sp3,
resulting in greater availability of Sp1 (Fig. 7). The viral
protein Nef also activates signaling cascades that result in
enhanced binding of AP-1 to the LTR and enhanced coop-
eration between AP-1 and NF-κB [210,214].
Although macrophages support active viral replication,
they are recognized as reservoirs of HIV-1 and quietly har-
bor the virus during latency. Host proteins that contribute
to LTR activation in macrophages during productive viral
infection ironically may also contribute to transcriptional
silencing during latency. Sp1 proteins have been shown to
bind the LTR constitutively, regardless of the level of tran-
scription [297], and, in the latent stage, Sp1, NF-κB, AP1,
and ATF/CREB may function as repressors of transcription
by recruiting HDACs to the LTR and promoting the forma-
tion of heterochromatin. Although AP-1 proteins become
constitutively expressed, the level of Ref-1, which is
required for the DNA binding activity of AP-1, is signifi-
cantly reduced in the nucleus of MDMs [201,202]. This
effectively renders nuclear AP-1 proteins inactive. In addi-

tion to their inability to transactivate the LTR, the consti-
tutive presence of AP-1 proteins may be sufficient to
disrupt the binding of C/EBP to the LTR, because inactive
heterodimers of AP-1 and C/EBP may be more likely to
form in the presence of excess AP-1 proteins. It is currently
unknown what triggers the switch from latency to produc-
tive replication, however the presence of factors that can
serve as both activators and repressors at the LTR likely
contributes to the ability of the virus to resume replication
very quickly upon the removal of repressive stimuli such
as HAART therapy.
Genetic variation within the LTR also plays a role in HIV-
1 transcription as HIV-associated disease progresses. Pre-
vious studies have shown that Vpr binds with high affinity
to specific configurations of sequences within the HIV-1
LTR C/EBP site I and NF-κB site II, and may directly acti-
vate transcription. The HIV-1 LTR C/EBP-NF-κB genotypic
configuration that exhibits high affinity for Vpr and low
affinity for C/EBPβ is prevalent during late stage HIV/
AIDS and in LTRs preferentially encountered in autopsied
brain tissue from individuals with HAD at the time of
death as compared to that from individuals without HAD.
In parallel with these observations, additional studies
have identified specific variants of the viral transactivator
Tat from HAD brain tissue that are defective with respect
to their ability to transactivate the LTR, but still retain the
ability to activate promoters of a number of proinflamma-
Retrovirology 2009, 6:118 />Page 14 of 24
(page number not for citation purposes)
tory cytokine genes [298]. In some tissues, such as the

brain, Tat may become less transcriptionally competent as
HIV-associated disease progresses. In these circumstances,
it is postulated that Vpr facilitates HIV-1 replication by
transactivation of LTR-directed transcription in the
absence of a fully active Tat protein.
Future directions
Transcription of the HIV-1 LTR is a highly complex proc-
ess that involves the interplay of host and viral transcrip-
tion factors coupled with a wide array of signaling
pathways that are activated by extracellular stimuli. Tar-
geting transcriptional pathways in drug discovery recently
proved effective in treating certain cancers and may pro-
vide an opportunity for additional therapeutic agents in
the highly active retroviral therapy (HAART) repertoire.
Stat3 has been declared "one of the most important onco-
genic transcription factors against which a targeted ther-
apy is needed" [299]. Constitutive Stat3 activity has been
observed in many cancers, including prostate [300], squa-
mous cell [301], breast [302,303], head, and neck cancers
[302], and has been associated with a poor prognosis
Cytokine-regulation of HIV-1 transcription in monocytesFigure 6
Cytokine-regulation of HIV-1 transcription in monocytes. Cytokines play an integral role in regulating the availability
and activity of transcription factors that regulate the LTR. TNFα strongly induces the nuclear localization of NF-κB in mono-
cytes. As a result, the subsequently stimulated LTR interfaces with increased levels of Sp and NF-κB factors. Cellular activation
increases the expression of C/EBP, particularly activation by IL-6. TNF-α, IL-1, and interferon-γ reduce the expression of C/
EBPα and increase expression of both C/EBPβ and C/EBPδ. Stimulation increases the expression of AP-1 in the cell where its
interaction with NF-κB at the enhancer element leads to synergistic activation of the LTR. (Black arrows: translocation to
nucleus; red arrows: decrease in expression; green arrows: increase in expression).
Retrovirology 2009, 6:118 />Page 15 of 24
(page number not for citation purposes)

[300]. c-Myc activity has been implicated in prostate can-
cer, melanoma, and Burkitt's lymphoma, and an anti-myc
antisense oligonucleotide has made it to clinical trials for
the treatment of prostate cancer [304]. Transcription fac-
tors that play critical roles in the regulation of HIV-1,
including NF-κB and Sp factors, are also the target of anti-
cancer drug development. NF-κB has been implicated in
playing a role in tumorigenesis in a variety of cancers
[305,306], including colon [307], prostate, breast, and
lung [308,309]. Small molecule inhibitors that target NF-
κB are currently under development for the treatment of
cancers [305], and have shown promise in small animal
models [310,311]. Many of these inhibit IκB phosphor-
ylation, resulting in NF-κB being sequestered in the cyto-
plasm [312]. Bortezomib was recently approved by the
FDA for the treatment of multiple myeloma. Developed as
a reversible 26S proteasome inhibitor, it is now believed
that its antitumor activity may be attributable to its inhi-
bition of NF-κB [313-315]. Tolfenamic acid, a nonsteroi-
dal anti-inflammatory drug approved for the treatment of
migraine headaches, has been shown to inhibit pancreatic
cancer cell growth in vitro and pancreatic and esophageal
tumor growth in vivo by inducting the proteosomal deg-
radation of Sp factors [316-320]. It also has been shown
Regulation of HIV-1 transcription in differentiated macrophagesFigure 7
Regulation of HIV-1 transcription in differentiated macrophages. In differentiated macrophages, NF-κB and NFAT are
constitutively localized in the nucleus; however, in the presence of large amounts of NF-κB, NFAT is unable to bind the LTR.
NF-κB-Sp1 protein-protein interactions bind the LTR cooperatively and activate transcription synergistically in response to cel-
lular stimuli. Sp sites are necessary for viral replication, and the ratio of Sp1 proteins to Sp3 proteins increases, thus increasing
transcription of the virus. As the cell matures, C/EBPα levels decrease and C/EBPβ and C/EBPδ levels increase. AP-1 is consti-

tutively expressed but loses its ability to bind to the LTR. Tat binds to the transactivation response region (TAR) structure on
the viral RNA and recruits (P-TEFb (the Cyclin dependent kinase 9 (Cdk9) and cyclin T1 (CycT1) complex) through binding to
cyclin T1. Recruitment of P-TEFb to TAR induces hyperphosphorylation of CTD by Cdk9, thereby enhancing the transcrip-
tional elongation of HIV-1.
Retrovirology 2009, 6:118 />Page 16 of 24
(page number not for citation purposes)
to decrease AP-2 and YY-1 transcription factor expression
in breast cancer cells and tumors [320]. P-TEFb has been
a target of chemotherapies for the treatment of renal, gas-
tric, and lung cancers, as well as mantle-cell lymphoma,
however clinical trials revealed that drugs targeting this
factor were not effective as monotherapies but showed
some promise when combined with other treatments
[321-325]. Drugs targeting P-TEFb have been shown to
inhibit HIV-1 transcription and replication in a dose-
dependent manner in cell lines with minimal cytotoxicity,
however the drugs were less effective and more cytotoxic
in primary PBMCs [326]. Further study is necessary to
determine the feasibility of applying other chemothera-
peutic drugs that target host transcription factors to
HAART therapy with important components of the devel-
opmental pathway focused on minimizing toxicity.
Vpr and Tat provide obvious candidates for targeted drug
therapy directed against HIV. Inhibition of Vpr-mediated
nuclear import by the compound hematoxylin has been
shown to decrease viral replication [327], and fumagillin
has been shown to suppress HIV-1 infection of macro-
phages by targeting Vpr-mediated growth arrest and tran-
scriptional activity [328]. Peptide analogs of Tat have
been shown to inhibit Tat's ability to recruit cdk2 to the

LTR, and to decrease transcription in vitro and viral load
in a small animal model of HIV-1 infection [329]. Small
molecular inhibitors have also been developed that dis-
rupt the Tat-TAR interaction, however these have not
developed into clinical trials [330-333]. In addition to tar-
geting individual viral proteins, unique structural motifs
created at the interface between these factors and host
transcription factors should also be considered in future
studies.
Abbreviations
AIDS: acquired immunodeficiency syndrome; AP-1: acti-
vator protein 1; ATF/CREB: activating transcription factor/
cyclic AMP response element-binding; bZIP: basic leucine
zipper; C/EBP: CCAAT enhancer binding protein; cdk9:
cyclin-dependent kinase 9; HAD: HIV-1-associated
dementia; HDACs: histone deacetylases; HIV: human
immunodeficiency virus; HIV-1: human immunodefi-
ciency virus type 1; HIV-2: human immunodeficiency
virus type 2; IL-1: interleukin-1; LTR: long terminal repeat;
MDMs: monocyte-derived macrophages; NFAT: nuclear
factor of activated T cells; NF-κB: nuclear factor kappa B;
NRE: negative regulatory element; SIV: simian immuno-
deficiency virus; Sp: stimulatory protein; TNFα: tumor
necrosis factor α.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
EK was responsible for drafting and revising the manu-
script as well as organizing the content. SS created Figures
1, 2, 3, 4, 5, 6, 7 and their legends and proofread the final

version of the manuscript for content and consistency.
MN drafted portions of the manuscript, assisted in the
conceptualization of the figures, and proofread and edited
the final version of the manuscript. BW assisted in all
aspects of each phase of development from initial con-
cept, through revisions to final approval of the version to
be published.
Acknowledgements
These studies were funded in part by the Public Health Service, National
Institutes of Health through grants (B. Wigdahl, Principal Investigator) from
the National Institute of Neurological Disorders and Stroke, NS32092 and
NS46263, and the National Institute of Drug Abuse, DA19807.
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